Disrupted iron homeostasis causes dopaminergic neurodegeneration in mice Pavle Mataka, Andrija Mataka, Sarah Moustafaa, Dipendra K. Aryalb,c,d,e, Eric J. Bennerf, William Wetselb,c,d,e, and Nancy C. Andrewsa,f,1 aDepartment of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC 27705; bDepartment of Psychiatry and Behavioral Sciences, Duke University School of Medicine, Durham, NC 27705; cDepartment of Cell Biology, Duke University School of Medicine, Durham, NC 27705; dDepartment of Neurobiology, Duke University School of Medicine, Durham, NC 27705; eMouse Behavioral and Neuroendocrine Analysis Core Facility, Duke University School of Medicine, Durham, NC 27705; and fDepartment of Pediatrics, Duke University School of Medicine, Durham, NC 27705 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2015. Contributed by Nancy C. Andrews, January 27, 2016 (sent for review September 30, 2015; reviewed by Michael K. Georgieff and Barry H. Paw) Disrupted brain iron homeostasis is a common feature of neuro- offers a very sensitive approach to detect cellular iron deficiency degenerative disease. To begin to understand how neuronal iron and surfeit (12). handling might be involved, we focused on dopaminergic neurons Iron homeostasis is altered locally, in the affected part of the and asked how inactivation of transport proteins affected iron brain, in most human neurodegenerative disorders (13). Iron ac- homeostasis in vivo in mice. Loss of the cellular iron exporter, cumulates in the substantia nigra (SN) in Parkinson’s disease (PD) ferroportin, had no apparent consequences. However, loss of (14–16), although it is unsettled whether increased iron is in neu- transferrin receptor 1, involved in iron uptake, caused neuronal ropil (17) or dopaminergic (DA) neurons (18), or both. Excess DA iron deficiency, age-progressive degeneration of a subset of dopami- neuron iron has been proposed to contribute to disease pathogen- nergic neurons, and motor deficits. There was gradual depletion of esis (19). Conversely, several reports suggest that systemic iron dopaminergic projections in the striatum followed by death of deficiency, rather than iron overload, may predispose to PD. PD has dopaminergic neurons in the substantia nigra. Damaged mitochon- been associated with a history of anemia years before the onset of dria accumulated, and gene expression signatures indicated attempted motor symptoms (20), with multiple blood donations that deplete axonal regeneration, a metabolic switch to glycolysis, oxidative stress, iron stores (21), and with low serum iron (22). Genetic pre- and the unfolded protein response. We demonstrate that loss of dispositiontoironoverloadappearstobeprotective(23–25). Thus, transferrin receptor 1, but not loss of ferroportin, can cause neuro- iron deposition in the SN may or may not be in DA neurons degeneration in a subset of dopaminergic neurons in mice. themselves, and iron overload, iron deficiency, or both might be iron | transferrin receptor | ferroportin | dopaminergic neuron | deleterious to DA neurons. neurodegeneration We used conditional KO mice to study how perturbations of iron transport affect DA neuron survival and function. Others pre- viously suggested that inactivation of FPN results in iron accumu- n the brain, iron is needed for mitochondrial respiration and lation in DA neurons, causing PD (19). However, it was not clear Isynthesis of myelin, neurotransmitters, and monoamine oxidases (1, 2). Transferrin receptor 1 (TFR1) is known to be required for iron uptake by some, but not all, cell types (3–7). Its ligand, trans- Significance ferrin (TF), carries extracellular iron. Fe2-TF/TFR1 is internalized by receptor-mediated endocytosis and trafficked to endosomes, The brain requires iron for mitochondrial respiration and synthesis where low pH liberates iron from TF, allowing it to leave the of myelin, neurotransmitters, and monoamine oxidases. Iron ac- endosome through transmembrane transport. This transport is cumulates in distinct parts of the brain in patients with neurode- mediated by divalent metal transporter 1 (DMT1/SLC11A2) in generative diseases, and some have proposed that neurons die erythroblasts and possibly other cell types (8). Imported iron is used because they contain too much iron. Neuronal iron handling is not directly, incorporated into heme or Fe-S clusters, or stored in fer- well understood. We focused on dopaminergic neurons, affected ritin. It has been widely assumed that neuronal iron uptake depends in Parkinson’s disease, and manipulated molecules involve in iron uptake and release. We showed that loss of ferroportin, which on TFR1-mediated endocytosis of Fe2-TF (9), but in vivo evidence is scant. Expression of a dominant negative form of Tfr1 was shown exports cellular iron, had no apparent effect. In contrast, loss of to decrease iron uptake by hippocampal CA1 pyramidal neurons transferrin receptor, involved in iron uptake, caused neuronal iron (10). However, others noted that iron distribution does not corre- deficiency and neurodegeneration with features similar to late with Tfr1 expression in the brain and suggested that Tfr1 may Parkinson’s disease. We propose that neuronal iron deficiency have roles unrelated to iron uptake (11). may contribute to neurodegeneration in human disease. There is only one known mechanism for cellular iron release, involving the transmembrane transporter ferroportin (FPN). FPN Author contributions: P.M., E.J.B., W.W., and N.C.A. designed research; P.M., A.M., S.M., and D.K.A. performed research; W.W. contributed new reagents/analytic tools; P.M., A.M., D.K.A., exports iron, in collaboration with ceruloplasmin or a related E.J.B., W.W., and N.C.A. analyzed data; and P.M., W.W., and N.C.A. wrote the paper. ferroxidase, to be loaded onto TF (8). Intracellular iron homeo- Reviewers: M.K.G., University of Minnesota; and B.H.P., Brigham & Women’s Hospital, stasis is controlled by iron-regulatory proteins (IRPs), which rec- Harvard Medical School. ognize iron regulatory elements (IREs) in the untranslated The authors declare no conflict of interest. portions of mRNAs encoding proteins important for iron trans- Freely available online through the PNAS open access option. port or storage (12). In iron deficiency, IRPs bind IREs to block Data deposition: The microarray data reported in this paper have been deposited in the ribosomal entry onto ferritin mRNAs, precluding translation of Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. this iron storage protein, and IRPs stabilize mRNAs encoding GSE66730). TFR1 and SLC11A2, facilitating iron uptake. In iron surfeit, fer- See Commentary on page 3417. ritin mRNAs are actively translated to store excess iron and iron 1To whom correspondence should be addressed. Email: [email protected]. import-related mRNAs are degraded. This well-studied homeo- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. static mechanism allows cells to regulate their iron content and 1073/pnas.1519473113/-/DCSupplemental. 3428–3435 | PNAS | March 29, 2016 | vol. 113 | no. 13 www.pnas.org/cgi/doi/10.1073/pnas.1519473113 Downloaded by guest on October 2, 2021 why DA neurons would require active iron export or why com- (Fig. 1B). Neurophysiological and behavioral tests (29) showed no pensatory changes in cellular iron import or storage would not differences at 16 mo. Th staining in the dorsal striatum and cell INAUGURAL ARTICLE maintain iron homeostasis. Accordingly, we found that mice lack- counts in the SN pars compacta (SNpc) were also indistinguish- ing Fpn in DA neurons had no apparent abnormalities in neuronal able at 19 mo (Fig. 1C). iron homeostasis and no evidence of neurodegeneration or DA Because the ventral midbrain contains multiple cell types, we neuron dysfunction. However, mice lacking Tfr1 in DA neurons could not use tissue to examine the iron content of DA neurons. developed a severe phenotype characterized by DA neuron iron We attempted to quantify iron content of DA neurons isolated insufficiency, progressive degeneration of neurons in the SN, de- from the ventral midbrain by FACS followed by inductively creased motor activity, and early death. We conclude that Tfr1 is coupled plasma MS (ICP-MS), but the amounts were too low. important for iron homeostasis in DA neurons of the SN and that Similarly, there was too little iron to visualize with Perls stain. As Fpn is dispensable. an alternative approach, we examined polysome-associated DA SEE COMMENTARY neuron mRNA for evidence of posttranscriptional regulation by Results the IRE/IRP system. Cellular iron surfeit should result in in- Deletion of Fpn in DA Neurons Has No Apparent Consequences. We creased Fth and Ftl and decreased Slc11a2 and Tfr1 mRNAs, but inactivated Fpn in DA neurons by crossing dopamine transporter we observed no significant differences between Fpn-CKO and (Dat)IRES Cre (26) and Fpnfl/fl mice (27) to obtain Fpn conditional CTR mice at 3 wk and 18 mo (Fig. 1D). We conclude that Fpn KO (Fpn-CKO) mice. We confirmed Fpn depletion by analyzing was not essential for DA neuron survival or function and Fpn- CKO DA neurons did not accumulate excess iron. polysome-associated mRNAs from ventral midbrain DA neurons. IRES To do this, we bred the floxed Fpn allele and Dat Cre transgene Tfr1 Is Critical for DA Neuron Iron Homeostasis, Function, and onto a Ribotag background (28) to allow for Cre-dependent ex- Survival. To determine whether Tfr1 was involved in DA neuron pression of hemagglutinin (HA)-tagged ribosomal protein L22. iron assimilation, we crossed Tfr1fl/fl and DatIRES Cre mice to We used the HA tag to immunoprecipitate polysome-associated obtain Tfr1-CKO animals. Ribotag polysome-associated mRNA mRNA from these mice at 3 wk and 18 mo of age and confirmed from ventral midbrain at 3 and 10 wk was highly enriched for DA that it was highly enriched for DA neuron transcripts and depleted neuron transcripts (Fig. S1 E–H) and depleted of Tfr1 mRNA of an oligodendrocyte marker (Fig.